Composite

Part:BBa_K3105680

Designed by: Jonas Gockel   Group: iGEM19_Uppsala_Universitet   (2019-10-12)
Revision as of 23:10, 19 October 2019 by JonasG (Talk | contribs)


Expression construct AAO-2A-eGFP

Expression circuit for aryl-alcohol oxidase (AAO) and eGFP in Pichia pastoris. AAO will be secreted due to the addition of the α-factor secretion signal and eGFP will remain in the cell. The 2A-selfcleaving peptide will lead to cleavage of the polypeptide, achieving separation of AAO from eGFP.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1356
    Illegal BamHI site found at 1543
    Illegal XhoI site found at 1183
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 2503
    Illegal AgeI site found at 3896
  • 1000
    COMPATIBLE WITH RFC[1000]



Characterization

The constructs was transformed in Pichia pastoris and colonies were screened on BMMY-agar plates (contain methanol for induction of expression) (Figure 1). Visualisation of eGFP by excitation with blue light shows eGFP positive clones. Those clones are logically also expressing aryl-alcohol oxidase, as described in the part description.

Figure 1: eGFP coexpression in 2A-constructs
Transformed P. pastoris cells coexpressing eGFP and AAO were screened on a BMMY agar plate and eGFP was visualised on a blue light box under an orange filter. eGFP activity is visible for two of the restreaked colonies, which are transformed with pPICZαB_AAO-2A-eGFP. Picture was taken after 4 days of incubation at 28 °C after daily addition of 50 µL methanol on the plate lid.




































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